甲胎蛋白启动子驱动的yCDglyTK双自杀基因治疗裸鼠肝癌的机制研究

目的研究携带甲胎蛋白（AFP）启动子的酵母菌胞嘧啶脱氨酶胸苷激酶（yCDglyTK）双自杀基因体内靶向性治疗肝癌的效果和机制。方法构建携带AFP启动子的yCDglyTK双自杀基因表达质粒,通过阳离子脂质体将携带AFP启动子的yCDglyTK双自杀基因转染HepG2和SMMC7721肝癌细胞的裸鼠肝癌皮下种植瘤,观察自杀基因体内杀瘤效果以及细胞凋亡的情况。结果成功构建携带AFP启动子的yCDglyTK双自杀基因,其靶向性地在AFP阳性的HepG2细胞种植瘤上表达,而AFP阴性的SMMC7721细胞种植瘤上无表达,氟胞嘧啶（5-FC）、更昔洛韦（GCV）及两者联合可有效抑制HepG2细胞种植瘤的生长,抑瘤效果GCV＋5-FC〉5-FC〉GCV,而SMMC7721细胞种植瘤的生长未受影响。HepG2细胞种植瘤治疗后有明显的细胞凋亡,而SMMC7721细胞种植瘤内极少凋亡细胞。结论携带AFP启动子的yCDglyTK双自杀基因能有效地靶向性地杀伤AFP阳性的肝癌细胞,细胞凋亡可能是其杀伤的重要机制之一。     

稳定表达Angiopoietin-1基因肝癌细胞株的建立及其意义

目的 通过将Angipoietin-1基因转染培养人肝癌细胞SMMC－7721,建立长期表达Angipoietin-1基因的肝癌细胞模型。方法 基因重组构建Angipoietin-1真核表达质粒pcDNA3/Angipoietin-1,经脂质体将pcDNA3/Angipoietin-1质粒转染培养人肝癌细胞株SMMC－7721,G418筛选阳性细胞克隆,逆转录－聚合酶链反应（RT－PCR）检测转染后25-30d细胞Angipoietin-1基因mRNA表达水平。结果 （1）限制酶切和凝胶电泳证明成功构建表达质粒pcDNA3／Angipoietin-1;（2）通过脂质体Dosper将质粒pcDNA3／Angipoietin-1转染SMMC－7721,经G418筛选后获得阳性细胞克隆;（3）RT－PCR证明经脂质体转染的人肝 癌细胞株SMMC－7721可较稳定表达Angipoietin-1基因。结论 成功建立稳定表达Angipoietin-1基因的肝癌细胞株SMMC－7721／Angipoietin-1,为通过在体途径探讨Angipoietin-1对肿瘤血管生成的调节作用奠定了基础。     

AIBP在肝癌细胞中的表达及意义与含AIBP基因及AFP启动子驱动的双自杀基因表达载体构建

目的：探讨载脂蛋白A-I结合蛋白（AIBP）在肝癌细胞中的表达及意义。方法：用RT-PCR与Western blot法分别检测正常肝细胞株L02,AFP阳性人肝癌细胞株HepG2和Hep3B,及AFP阴性人肝癌细胞株SMMC7721中AIBP基因与蛋白的表达;构建AFP启动子驱动的双自杀基因（CD,TK）+AIBP基因过表达载体pcDNA3.1-AFP-AIBP-yCD/TK,并转染Hep3B和SMMC7721细胞,用MTT法检测转染后细胞的增殖能力,用RT-PCR和Western blot法检测细胞AIBP,血管内皮生长因子（VEGF）,VEGF受体2（VEGFR-2）,基质金属蛋白酶9（MMP-9）基因和蛋白的表达。结果：AIBP mRNA和蛋白在正常肝细胞中高表达,而在各肝癌细胞系中均表达下调,且Hep3B和SMMC7721细胞中下调明显。成功构建pcDNA3.1-AFP-AIBP-yCD/TK真核表达质粒并转染入Hep3B和SMMC7721细胞。转染后AFP阳性Hep3B细胞生长到明显抑制,但AFP阴性SMMC7721细胞增殖不受影响;两种细胞的AIBP基因与蛋白表达均明显上调,而VEGFR-2,VEGF和MMP-9基因与蛋白表达明显表达下调。定量指标间的差异均有统计学意义（均P<0.05）。结论：AIBP在肝癌细胞中表达下调,AIBP与肝癌细胞的侵袭转移能力有关,而与细胞增殖能力无关;成功构建了联合基因载体pcDNA3.1-AFP-AIBP-yCD/TK。

     

TRAIL的抗肝细胞癌作用研究

目的 探讨TRAIL对HCC的治疗作用。方法 用不同浓度TRAIL处理肝癌细胞株HepG2、SMMC7721，观察经药物处理前后肿瘤细胞的凋亡发生率。采用分子克隆技术构建了真核表达质粒pIRES-EGFP-TRAIL，转染肝癌细胞株HepG2、SMMC7721细胞，观察其疗效。体内实验建立裸鼠肝癌模型，观察TRAn。的抑癌作用。结果TRAR，(100ng／m1)处理24h，肝癌细胞凋亡发生率约10％，而Jurkat细胞凋亡率达70％以上，胆管癌细胞QBC939凋亡发生率约50％。真核表达质粒TRAIL体外转染肝癌细胞后对肝癌细胞的生长无显著性的抑制作用。体内直接瘤体注射pIRES-EGFP-TRAIL可有效的导入TRAIL基因并获得高表达，但对裸鼠肝癌无明显抑制作用。结论 肝细胞癌对TRAIL诱导的凋亡有耐药现象，提示HCC中存在抑制TRAIL诱导凋亡的因素，单一的TRAIL疗HCC疗效有限。     

RNA干扰技术靶向SMYD3基因治疗肝癌的实验研究

目的 构建针对人SET-和MYND-结构域含有蛋白3（SMYD3）编码基因的短发夹状RNA（shRNA）干扰质粒,并研究其对肝癌细胞的作用。方法 应用逆转录聚合酶链反应（RT-PCR）方法检测SMYD3mRNA在肝癌细胞系HepG2、Hep3B、SMMC7721的表达。构建重组SMYD3shRNA表达质粒Pgenesil-1-s,并采用介导转染法导入HepG2细胞,蛋白免疫印迹法（Western blot）检测阻抑效应,噻唑蓝比色法（MTT）检测细胞生长增殖抑制率,流式细胞术检测细胞凋亡率。建立人肝癌细胞HepG2皮下移植瘤裸鼠模型,注射Pgenesil-1-s,动态观察肿瘤体积并于2周后处死裸鼠称取瘤重。结果肝癌细胞株HepG2、Hep3B、SMMC7721的SMYD3mRNA表达水平显著高于正常肝细胞株L-02;Pgenesil-1-s转染HepG2细胞后,SMYD3蛋白表达明显下调,而且细胞凋亡率显著增加,细胞生长明显受抑;经重组质粒治疗14d后,裸鼠皮下移植瘤生长缓慢,相较于对照组瘤体明显缩小、瘤重明显减轻（P〈0．01）。结论 肝癌细胞高表达SMYD3,shRNA干扰特异性抑制SMYD3表达,可以促进肝癌细胞凋亡并抑制裸鼠移植瘤的生长。     

放射线敏感性自杀基因体外杀伤肝癌细胞的实验研究

目的观察可被放射线转录激活的早期生长反应基因 1(earlygrowthresponse 1,Egr 1)启动子驱动的单纯疱疹病毒胸苷激酶基因 (herpssimplexvirusthymidinekinase ,tk)对肝癌细胞的高效杀伤作用。方法构建以Egr 1为启动子 ,以tk为目的基因的重组质粒pET ,经脂质体介导转染人肝癌细胞株SMMC 772 1,命名为SMMC/ET细胞 ,经G418抗性筛选、60 Co γ射线照射后 ,加入前药丙氧鸟苷 (ganciclovirGCV) ,测定其对肝癌细胞的杀伤作用。结果与对照组肝癌细胞 ( 0 88± 0 12 )相比 ,经γ射线照射后 ,前药GCV可明显提高对SMMC/ET肝癌细胞的杀伤效率 ( 0 0 7± 0 0 3) (P <0 0 0 1)。结论放射敏感性自杀基因在γ射线作用下可以显著提高对肝癌细胞的杀伤作用。     

体外腺病毒介导的HSV-tk/GCV对产生AFP的人肝癌细胞的影响

目的 观察腺病毒介导的单纯疱疹病毒胸苷激酶基因（HSV-tk）/丙氧鸟苷（GCV）自杀基因系统在体外对人肝癌细胞的选择性杀伤效应。方法 采用含有甲胎蛋白基因启动子、增强子和HSV-tk基因的嵌合基因,插入腺病毒中形成重组腺病毒（AdrAFPTK）,将该重组腺病毒分别感染体外培养的甲胎蛋白阳性人肝癌细胞BEL-7402和甲胎蛋白阴性人肝癌细胞SMMC-7721,逆转录聚合酶链式反应（RT-PCR）检测HSV-tk基因的转录表达,观察GCV对人肝癌细胞的选择性杀伤作用。结果GCV在体外对重组腺病毒转染的甲胎蛋白阳性的人肝癌细胞BEL-7402有明显的杀伤作用和“旁观者效应”,而对重组腺病毒转染的甲胎蛋白阴性的人肝癌细胞SMMC-7721无明显作用。结论 在体外,表达HSV-tk基因的甲胎蛋白的人肝癌细胞可被受甲胎蛋白基因表达调控序列控制的自杀基因HSV-tk特异性杀伤,表现出极高的细胞专一性。重组腺病毒AdrAFPTK可望用于肝癌的特异性基因治疗。     

体外腺病毒介导的HSV—tk／GCV对产生AFP的人肝癌细胞的影响

目的 观察腺病毒介导的单纯疱疹病毒胸苷激酶基因（HSV-tk)/丙氧鸟苷（GCV）自杀基因系统在体外对人肝癌细胞的选择性杀伤效应。方法 采用含有甲胎蛋白基因启动子，增强子和HSV-tk基因的嵌合基因，插入腺病毒中形成重组腺病毒（AdrAFPTK),将该重组腺病毒分别感染体外培养的甲胎蛋白阳性人肝癌细胞BEL-7402和甲胎蛋白阴性人肝癌细胞SMMC-7721，逆转录聚合酶链式反应（RT-PCR）检测HSV-tk基因的转录表达，观察GCV对人肝癌细胞的选择性杀伤作用。结果 GCV在体外对重组腺病毒转染的甲胎蛋白阳性的人肝癌细胞BEL-7402有明显的杀伤作用和“旁观效应”，而对重组腺病毒转染的甲胎蛋白阴性的人肝癌细胞SMMC-7721无明显作用。结论 在体外，表达HSV-tk基因的甲胎蛋白的人肝癌细胞可被受甲胎蛋白基因表达调控序列控制的自杀基因HSV-tk特异性杀伤，表现出极高的细胞专一性，重组腺病毒AdrAFPTK可望用于肝癌的特异性基因治疗。     

靶向IGFIR的siRNA抑制人肝癌裸鼠移植瘤的实验研究

目的:探讨人胰岛素样生长因子1类受体（IGFIR）干扰质粒(PSUPER-siRNA-IGFIR)对人肝癌细胞株SMMC7721裸鼠移植瘤生长的抑制作用。方法:建立人肝癌细胞株SMMC7721裸鼠移植瘤模型，将PSUPER-siRNA-IGFIR质粒转染入移植瘤中，观察对肿瘤生长的作用。结果:PSUPER-siRNA-IGFIR对人肝癌细胞株SMMC7721裸鼠移植瘤有生长抑制作用。与对照组相比IGFIR和MVD的表达明显下降。结论: PSUPER-siRNA-IGFIR能抑制肝癌裸鼠移植瘤的生长。     

TK自杀基因对甲胎蛋白阳性肝癌的特异性杀伤作用

目的观察腺病毒介导的单纯疱疹病毒胸苷激酶基因(HSV-tk)/丙氧鸟苷(GCV)自杀基因系统在体内外对人肝癌细胞的杀伤效应.方法在体外按MOI值为100、10、1、0用重组腺病毒转染人肝癌细胞BEL-7402和SMMC-7721,48 h后用噻唑蓝(MTT)法测定细胞存活率.在两种肝癌裸鼠模型上,瘤体内注射重组腺病毒(1×1012 pfu/L)0.1 ml,GCV作用后观察抑瘤效果.结果体外MOI为100时,可杀死99.8%的BEL-7402细胞和17.8%的SMMC-7721细胞,两者差异有显著性(P＜0.05).体内BEL-7402组肿瘤生长明显受到抑制.结论在体内外,腺病毒介导的含AFP调控序列的HSV-tk/GCV自杀基因系统对甲胎蛋白(AFP)阳性肝癌细胞具有特异性杀伤作用,该系统可望用于肝癌的特异性基因治疗.     

人肝癌特异性 HSV-TK/GCV重组腺病毒相关病毒质粒的构建与研究

目的 构建肝癌特异性HSV—TK／GCV重组腺病毒相关病毒质粒并了解其在细胞内的表达。方法 以腺病毒相关病毒的质粒WAV2作为载体，将TK基因插在甲胎蛋白(AFP)增强子／白蛋白启动子(AFP增强子／ALB启动子)的调控基因的下游，重组成pWAV2／AFP-ALB／HYTK质粒载体。同时构建质粒载体pEGFP-1／AFP—ALB。然后将上述两种不同载体分别转入AFP阳性表达的HepG2细胞株以及阴性表达的7721、SPC和7901细胞株。结果 绿色荧光蛋白只在AFP阳性表达的HepG2细胞株表达，采用PCR技术，以HSV—TK的引物扩增所抽提的总DNA中，只有AFP阳性表达的HepG2细胞株扩增出了710bp的DNA片段。结论 pWAV2／AFP—ALB／HYTK质粒载体在体外实验中具有很好的靶向性。     

Prognostic Significance of Alpha-fetoprotein Status in the Outcome of Hepatocellular Carcinoma after Treatment of Transarterial Chemoembolization

Background

Alpha-fetoprotein (AFP) has been used as a diagnostic biomarker for hepatocellular carcinoma (HCC), but its prognostic significance is not well defined. This study was performed to classify the prognostic significance of AFP status in HCC patients after transarterial chemoembolization (TACE).

Methods

Four hundred forty-one HCC patients from a prospective maintained database with pathologic confirmation including 139 with normal AFP levels and 302 with elevated AFP levels were retrospectively studied for prognostic significance of AFP in treatment response and survival after TACE. Univariate and multivariate analyses were used to identify the prognostic factors.

Results

There were significant differences in overall survival (OS) after TACE between AFP-negative and AFP-positive HCC patients when the AFP cutoff value was defined as 20?ng/ml (P?P?=?0.093). Multivariate analysis revealed that AFP status for AFP-negative or positive was an independent prognostic factor for HCC patients after TACE (P?=?0.001), along with ??-glutamyltransferase (GGT) level (P?=?0.004) and tumor diameter (P?Conclusions Compared with AFP-positive HCC patients, patients with AFP-negative status have a better treatment response and prognosis after TACE.     

肝癌组织中Bcl-2和NF-κB的表达与AFP相关性的研究

目的：观察凋亡相关基因Bcl-2、核因子NF-κB在肝细胞性肝癌（HCC）组织中的表达,并探讨二者与AFP之间的关系。方法：采用免疫组织化学方法检测40例HCC组织（其中AFP阳性20例,AFP阴性20例）和10例良性肿瘤组织中Bcl-2、NF-κB的表达。结果：40例HCC患者Bcl-2阳性表达率为45.0%,NF-κB阳性表达率为57.5%;其中AFP阳性细胞Bcl-2表达率为55.0%,AFP阴性细胞为35.0%;AFP阳性细胞NF-κB表达率为75.0%,AFP阴性细胞为40.0%。HCC细胞NF-kB和Bcl-2的表达呈正相关（r=0.778,P〈0.01）。结论：Bcl-2、NF-κB的异常表达同HCC的抗凋亡和耐药性有一定关系,AFP阳性HCC细胞的Bcl-2、NF-κB阳性表达率更高。Bcl-2和NF-κB可以考虑作为预后的判断指标之一。     

肝癌上皮-间叶样表型转化与Caveolin-1表达之间的关系

目的 探讨肝细胞癌中Caveolin-1表达与上皮-间叶样表型转化(EMT)之间的关系.方法 应用逆转录-聚合酶链反应(RT-PCR)、Western blot和免疫组织化学技术检测人肝癌细胞株和肝细胞癌组织标本中Caveolin-1和EMT指标mRNA和蛋白的表达.结果 RT-PCR和Westernblot结果显示,在HepG2、SMMC7721细胞株中,Caveolin-1 mRNA表达量(0.03±0.01、26.63±0.49)和蛋白表达量(0.00 ±0.00、1.56 ±0.17)均与上皮细胞标志物E-cadherin(14.97 ±0.03、0.06±0.00)、(1.04±0.06、0.00±0.00)负相关,与间叶细胞标志物N-cadherin(2.03±0.07、9.54 ±0.05、0.68 ±0.09、0.95±0.05)、Vimentin(0.13±0.06、6.13±0.68、0.00±0.00、1.09±0.15)正相关.免疫组织化学结果显示,在82例肝细胞癌组织标本中,转移组癌组织中Caveolin-1表达明显低于癌旁组织(P<0.05),且与上皮细胞标志物ZO-1、E-cadherin负相关,与间叶细胞标志物N-cadherin正相关.结论 肝细胞癌中Caveolin-1表达升高与转移和EMT有关.     

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??Construction and expression of enhanced green fluorescent protein and HSV1-TK co-expression vector TANG Yong , LIU Zuo-jin, ZHOU Shi-ji ,et al. Department of Hepatobiliary Surgery, the Second Affiliated Hospital, Chongqing University of Medical Scienses, Chongqing 400010??China Corresponding author: LIU Chang-an, E-mail??liuchanganyisheng@sina.com Abstract Objective To construct EGFP and HSV1-TK co-expression vector and to detect its expression level in hepatoma carcinoma cell line HepG2, and thus to provide experimented bases for the target gene therapy of liver cancer. Methods HSV1-TK gene was isolated from pORF-HSV1TK plasmids and cloned into eukaryotic expression plasmid pIRES2-EGFP??The recombinant plasmid pIRES2-EGFP-TK was identified by restriction endonuclease digestion and DNA sequencing. Then recombinant plasmid was transfected into hepatoma carcinoma cell line HepG2 by liposome reagent??and the expression of EFGP in cell were observed by fluorescence microscopy , TK protein and mRNA was analyzed by Western blotting and RT-PCR respectively?? Results The sequence of the cloned DNA fragment was identical to HSV1-TK that was reported on Gene bank??The recombinant expression plasmid was successfully transfected into HepG2 cell line, and green fluorescent was observed under fluorescent microscope, positive clones were picked out by G418 screening. The optimal G418 screening concentration was 600g/L.The mRNA expression level of TK was high and its protein expression was found. Conclusion The recombinant eukaryotic co-expression vector of EGFP and HSV1-TK was successfully constructed and effectively expressed in HepG2 cell line.     

Is Serum Alpha-Fetoprotein Useful for Predicting Recurrence and Mortality Specific to Hepatocellular Carcinoma After Hepatectomy? A Test Based on Propensity Scores and Competing Risks Analysis

Background

Serum alpha-fetoprotein (AFP) is frequently used to predict posthepatectomy outcomes in patients with hepatocellular carcinoma (HCC), but its predictive value is still not established. Therefore, we assessed the prognostic significance of AFP status.

Methods

Of 525 patients undergoing curative hepatectomy for HCC, 290 had preoperative AFP levels of ??20?ng/mL (AFP-positive group) and 235 had AFP levels of <20?ng/mL (AFP-negative group). We compared the 2 groups with respect to time-to-recurrence, using the inverse probability of treatment weighted (IPTW) for the entire cohort and propensity score matching, and the cumulative incidence of HCC-specific mortality using competing risks regression.

Results

During follow-up (median duration 64?months, range 2?C137?months), HCC recurred in 54.9?% of the AFP-negative group and 52.4?% of the AFP-positive group; there was no death without recurrence. After IPTW adjustment, time-to-recurrence did not differ in the 2 groups (hazard ratio [HR] 0.86, 95?% confidence interval [95?% CI] 0.66?C1.12; P?=?0.28). In a propensity-score matched cohort (152 pairs), time-to-recurrence data were similar to those obtained by IPTW adjustment (HR 0.91, 95?% CI 0.65?C1.25; P?=?0.55). There was no difference in recurrence pattern (site and stage) or treatment between the 2 groups even after propensity-score matching. The adjusted HR evaluating the impact of AFP positivity on the risk of HCC-specific mortality was 0.77 (95?% CI 0.54?C1.08; P?=?0.13) A multivariable competing risks analysis also failed to reveal a significant correlation between baseline AFP level and HCC-specific mortality in the AFP-positive group.

Conclusions

Preoperative AFP levels are not useful for predicting recurrence or survival endpoints following curative hepatectomy for HCC.     

不同侵袭能力人肝癌细胞株中caveolin-1的表达

目的:探讨不同侵袭能力人肝癌细胞株中caveolin-1的表达情况及意义.方法:应用免疫细胞化学、Western blot、RT-PCR和ELISA等技术检测人正常肝细胞株L02、肝癌细胞株HepG2和SMMC-7721中caveolin-1蛋白及mRNA的表达,同时检测各自血管内皮生长因子(vascular en-dothelial growth factor,VEGF)的表达.结果:caveolin-1蛋白和mRNA在正常肝细胞株中呈弱表达,在低侵袭肝癌细胞株中表达缺失,而在较高侵袭肝癌细胞株中表达显著上升,且与VEGF关系密切.结论:caveolin-1的表达与肝癌侵袭和血管生成有关,且在肝癌发生的不同阶段作用不同.